Lactuca sativa cultivar cvx-35

ABSTRACT

According to the invention, there is provided a novel romaine lettuce cultivar, designated CVX-35. ‘CVX-35’ is described as a vigorous romaine cultivar recommended for the main lettuce growing regions of California and Arizona. It has larger frame size and heavier weight, short core length, savoyed and glossy leaf color, corky root rot resistance, Bushy Stunt virus resistance, no fringe burn on mature leaves and also yellower heart leaf color. The present invention relates to a new and distinctive cos or romaine lettuce ( Lactuca sativa  L.) variety designated CVX-35. This invention thus relates to the seeds of lettuce cultivar CVX-35, to the plants of lettuce cultivar CVX-35, to plant parts of lettuce cultivar CVX-35, to methods for producing a lettuce cultivar by crossing the lettuce cultivar CVX-35 with another lettuce cultivar, and to methods for producing a lettuce cultivar containing in its genetic material one or more backcross conversion traits or transgenes and to the backcross conversion lettuce plants and plant parts produced by those methods.

CROSS REFERENCE TO RELATED APPLICATION

This is a Continuation Application of U.S. Ser. No. 15/085,277, filedMar. 30, 2016, which is herein incorporated by reference in itsentirety.

FIELD OF THE INVENTION

The present invention relates to the field of plant breeding. Inparticular, this invention relates to a new lettuce variety designated“CVX-35”.

BACKGROUND OF THE INVENTION

Lettuce is an increasingly popular crop. Worldwide lettuce consumptioncontinues to increase. As a result of this demand, there is a continuedneed for new lettuce varieties. In particular, there is a need forimproved romaine lettuce varieties that exhibit larger frame size andheavier weight, short core length, savoyed and glossy leaf color, corkyroot rot resistance, bushy stunt virus resistance, no fringe burn onmature leaves and also yellower heart leaf color. The present inventionrelates to a new and distinctive cos or romaine lettuce (Lactuca sativaL.) variety designated CVX-35. All publications cited in thisapplication are herein incorporated by reference.

Most cultivated forms of lettuce belong to the highly polymorphicspecies Lactuca sativa that is grown for its edible head and leaves.Lactuca sativa is in the Cichoreae tribe of the Asteraceae (Compositae)family. Lettuce is related to chicory, sunflower, aster, dandelion,artichoke and chrysanthemum. Sativa is one of about 300 species in thegenus Lactuca.

Presently, there are over a thousand known lettuce varieties withinseven different morphological types. The crisphead group includes theiceberg and batavian types. Iceberg lettuce has a large, firm head witha crisp texture and a white or creamy yellow interior. The batavianlettuce predates the iceberg type and has a smaller and less firm head.The butterhead group has a small, soft head with an almost oily texture.The romaine, also known as cos lettuce, has elongated upright leavesforming a loose, loaf-shaped head and the outer leaves are usually darkgreen. Leaf lettuce comes in many varieties, none of which form a head,and include the green oak leaf variety. The next three types are seldomseen in the United States: Latin lettuce looks like a cross betweenromaine and butterhead; stem lettuce has long, narrow leaves and thick,edible stems; and oilseed lettuce is a type grown for its large seedsthat are pressed to obtain oil.

The romaine group of lettuces is characterized by large, cylindrical,semi-firm heads averaging 30.0 cm in diameter and 800 g in weight, whichare borne on a set of frame leaves that form the base of the plant. Theheads are composed of leaves that are spirally arranged on a stem withgreatly foreshortened internodes, where the leaves are loosely claspingupon one another forming a roll of elongated, spatula-shaped (spatulate)leaves, where the length is normally 50% longer than the width, having arange of length to width ratios of 1.2 to 2.5, where 1.5 is most common.Romaine lettuces generally have a semi-open head formation. The name“romaine” comes from the French for “Roman”. Outer leaves range in colorintensity from dark green (RHS 146A) to mid-green (RHS 146B) to lightgreen (RHS 146C) with inner leaves ranging from green (RHS 146B) tolight green (RHS 146D). More information regarding the generalcharacteristics of romaine lettuce may be found in Ryder, E. J., LeafySalad Vegetables, AVI Publishing Company.

Romaine lettuce is Lactuca sativa L. var. longifolia Lam; also known asCos. The plant develops in an upright open or upright compact growinghabit with coarse textured leaves. The leaves are longer than they arewide, cupping together to form an elongated loose head. Leaf margins areoften entire or undulated, rarely frilled. Outer leaves range in colorfrom light green to dark green with a heavy midrib. Inner heart leavesare smaller and range from light yellow to light green in color.

Lettuce in general and leaf lettuce in particular is an important andvaluable vegetable crop. Thus, a continuing goal of lettuce plantbreeders is to develop stable, high yielding lettuce cultivars that areagronomically sound. To accomplish this goal, the lettuce breeder mustselect and develop lettuce plants with traits that result in superiorcultivars.

Problems with existing cultivars adapted to western conditions include alack of resistance to corky root rot. Corky root rot is believed to becaused by a pathogenic soil bacterium of the genus Rhizomonas. Onespecies of Rhizomonas that is commonly found to cause corky root rot isR. suberifaciens. Corky root rot accounts for significant lettuce croploss in the western United States, particularly in the valleys of thecentral coast of California, i.e., the Salinas, Santa Maria, and Lompocvalleys.

Corky root rot symptoms include yellow bands on tap and lateral roots oflettuce seedlings. Guide to Leafy Vegetable Production in the Far West,Ron Smith, ed., California-Arizona Farm Press (1997). Yellow areasgradually expand and develop a green-brown color with cracks and roughareas on the root surface. The entire taproot may become brown, severelycracked and may cease to function. Feeder root systems are reduced anddamaged. Roots become very brittle and break off easily. When the rootis severely discolored, above ground symptoms show up as wilting duringwarm temperatures, stunting and general poor, uneven growth. Loss of theroot system results in stunted plants that are chlorotic and too smallto harvest.

Additionally, color is a very important trait. Various shades of colorcan determine whether a food product is successful at market. A deepershade of green looks more appetizing than a pale shade of green or agreen with a yellowish tint. A deep green in a ripe, healthy head oflettuce is especially desirable in a Romaine lettuce and its varietiesand has been found to be especially strong commercially.

There are numerous steps in the development of any novel, desirableplant germplasm. Plant breeding preferably begins with the analysis anddefinition of problems and weaknesses of the current germplasm, theestablishment of program goals, and the definition of specific breedingobjectives. The next step is preferably selection of germplasm thatpossess the traits to meet the program goals. The goal is to combine ina single variety or hybrid an improved combination of desirable traitsfrom the parental germplasm.

For a further understanding of lettuce, its uses and history see Waycottet al, U.S. Pat. No. 5,973,232 and Subbaroa 1998, which are herebyincorporated by reference in their entirety.

The foregoing examples of the related art and limitations relatedtherewith are intended to be illustrative and not exclusive. Otherlimitations of the related art will become apparent to those of skill inthe art upon a reading of the specification.

SUMMARY OF THE INVENTION

According to the invention, there is provided a novel lettuce cultivar,designated CVX-35. This invention thus relates to the seeds of lettucecultivar CVX-35, to the plants of lettuce cultivar CVX-35, to plantparts of lettuce cultivar CVX-35, to methods for producing a lettucecultivar produced by crossing the lettuce cultivar CVX-35 with anotherlettuce cultivar, and to methods for producing a lettuce cultivarcontaining in its genetic material one or more backcross conversiontraits or transgenes and to the backcross conversion lettuce plants andplant parts produced by those methods. This invention also relates tolettuce cultivars and plant parts derived from lettuce cultivar CVX-35,to methods for producing other lettuce cultivars derived from lettucecultivar CVX-35 and to the lettuce cultivars and their parts derived bythe use of those methods. This invention further relates to lettucecultivar seeds, plants and plant parts produced by crossing the lettucecultivar CVX-35 or a backcross conversion of CVX-35 with another lettucecultivar.

DETAILED DESCRIPTION OF THE INVENTION Definitions

In the description and tables which follow, a number of terms are used.In order to provide a clear and consistent understanding of the presentinvention, the following definitions are provided:

Allele. The allele is any of one or more alternative forms of a geneticsequence. In a diploid cell or organism, the two alleles of a givengenetic sequence occupy corresponding loci on a pair of homologouschromosomes.

Backcrossing. Backcrossing is a process in which a breeder crossesprogeny back to one of the parents one or more times, for example, afirst generation hybrid F₁ with one of the parental genotype of the F₁hybrid.

Bolt. The process during which the stem within the lettuce head greatlyelongates, causing the head to lose its shape and resulting ultimatelyin the producing of a flowering stalk.

Butt. The bottom portion of the lettuce which includes the stem andadjacent leaf bases of the outermost head leaves.

Core. The stem of the lettuce head on which the leaves are borne.

Core Length. Length of the internal lettuce stem, measured from the baseof the cut head to the tip of the core.

Core Diameter. Diameter of the stem at the base of the cut head.

Core Value Coefficient. Calculated by taking the core length andmultiplied by diameter which compares the core shapes. The larger thecore volume coefficient value, the longer and narrower is the core.Inversely, the smaller the core volume coefficient number, the shorterand stubbier the core.

Cotyledon. In the case of lettuce, one of a pair of leaves formed on anembryo within a seed, which upon germination are the first leaves toemerge.

Essentially all the physiological and morphological characteristics. Aplant having essentially all the physiological and morphologicalcharacteristics means a plant having the physiological and morphologicalcharacteristics of the recurrent parent, except for the characteristicsderived from the converted gene.

First outer leaf. As described herein, “first outer leaf” means thefirst leaf located on the outer surface of the lettuce head.

First water date. The date the seed first receives adequate moisture togerminate. This can and often does equal the planting date.

Fourth Leaf. The fourth leaf formed on the lettuce plantlet subsequentto the emergence of the cotyledons.

Frame Diameter. A horizontal measurement of the plant diameter at itswidest point, from outer most leaf tip to outermost leaf tip.

Frame Leaf. The first set of freely recurring leaves which are externalto the head.

Head diameter. Diameter of the market cut and trimmed head with singlecap leaf.

Head weight. The weight of a marketable lettuce head, cut and trimmed tomarket specifications.

Leaf area coefficient. Comparison of leaf areas or size between multiplevarieties. This is calculated by multiplying the leaf width by the leaflength.

Leaf Index. Comparison of leaf shape between multiple varieties. This iscalculated by dividing the leaf length by the leaf width.

Maturity. Refers to the stage when the plants have mature head formationand are harvestable.

Rogueing. Process in lettuce seed production where undesired plants areremoved from a variety because they differ physically from the general,desired expressed characteristics of the new variety.

Plant Part. As used herein, the term “plant part” includes leaves,stems, roots, seed, embryos, pollen, ovules, flowers, root tips,anthers, tissue, cells, axillary buds, and the like.

Plant Cell. As used herein, the term “plant cell” includes plant cellswhether isolated, in tissue culture or incorporated in a plant or plantpart.

Plant Tissue Color Chart. Refers to the Munsell Color Chart for PlantTissue which publishes an official botanical color chart quantitativelyidentifying colors according to a defined numbering system. The MunsellColor Chart for Plant Tissue may be purchased from Munsell ColorServices, 617 Little Britain Road, Suite 102, New Windsor, N.Y.12553-6148, USA, Part Number: 50150.

Lettuce ‘CVX-35’ was originated in year 2000 from a manmade crossbetween two proprietary varieties. ‘CVX-35’ and is genetically uniformand stable and no genetic variants or off-types have been observed inthe last three generations.

‘CVX-35’ belongs to the romaine lettuce, Lactuca sativa L. varieties.‘CVX-35’ is described as a vigorous romaine cultivar and recommended forthe main lettuce growing regions of California and limited areas ofArizona with optimum sowing during the fall planting season.

‘CVX-35’ was bred and selected for larger frame size and heavier weight,short core length, savoyed and glossy leaf color, corky root rotresistance, bushy stunt virus resistance, no fringe burn on matureleaves and also yellower heart leaf color.

‘CVX-35’ is distinguishable to cv. Bondi based on the following:

According to the Munsell Color Chart for Plant Tissues, ‘CVX-35’ hasleaf color Hue 7.5 GY, Value 4 and Chroma 6, whereas cv. Bondi has leafcolor Hue 5 GY, Value 5 and Chroma 10.

A variety description of Lettuce Cultivar CVX-35 is provided in Table 1.

TABLE 1 Variety Description Information TRAIT Plant Type Cos or RomaineSEED Color Black (Grey Brown) Light dormancy Light Not Required Heatdormancy Susceptible COTYLEDON TO FOURTH LEAF STAGE Shape of CotyledonsBroad Shape Of Fourth Leaf Elongated Length/Width Index of Fourth Leaf19 (Length/Width × 10) Apical Margin Entire Basal Margin ModeratelyDentate Undulation Flat Green Color Medium Green AnthocyaninDistribution Absent Anthocyanin Rolling Absent Anthocyanin CuppingUncupped Anthocyanin Reflexing None MATURE LEAF Margin Incision DepthAbsent/Shallow (Dark Green (deepest penetration of the margin) Boston)Margin Indentation Entire (Dark Green Boston) (finest divisions of themargin) Undulations of the Apical Margin Absent/Slight (Dark GreenBoston) Green Color Very Dark Green Anthocyanin Distribution AbsentAnthocyanin Size Large Anthocyanin Glossiness Glossy (Great Lakes)Anthocyanin Blistering Absent/Slight (Salinas) Anthocyanin LeafThickness Thick Anthocyanin Trichomes Absent (Smooth) PLANT Spread ofFrame Leaves 17 cm Head Size Class Large Head Shape Non-Heading Head PerCarton 24 Head Weight 0654 grams BUTT Shape Rounded Midrib Flattened(Salinas) CORE Diameter at Base of Head 41 mm Core Height From Base ofHead to 78 mm Apex BOLTING Number of Days from First Water 75 Date toSeed Stalk Emergence (summer conditions) Bolting Class slow Height ofMature Seed Stalk 95 cm Spread of Bolter Plant 32 cm Bolter LeavesStraight Margin Entire Color Medium Green Bolter Habit TerminalInflorescence Present Bolter Habit Lateral Shoots Present Bolter HabitBasal Side Shoots Absent MATURITY Number of Days from First Water 66Date to Harvest (spring) Number of Days from First Water 70 Date toHarvest (summer) ADAPTATION Southwest and West Coast Adapted SeasonSpring(CA, AZ), summer (CA), fall (AZ) Soil Type Mineral and OrganicFUNGAL/BACTERIAL DISEASES Corky Root Rot Resistant Bushy Stunt VirusResistant

Table 2 below is the evaluation of ‘CVX-35’and the most similarcultivar, cv. Bondi, for several important field traits.

No. Plants/ t*- Avg. Variable Trial Rep value p [t*] ‘CVX35’ Avg. ‘BondiHead Weight (g) 1 20 10.5 0.000 686.0 ± 23.03 574.0 ± 44.17

 Confidence Interval   637 to 734.2   481 to 666.5 2 20 9.1 0.000 621.5± 43.9  511.0 ± 31.2  Confidence Interval 529 to 713 445 to 576 CoreDiameter (cm) 1 20 3.4 0.002  4.1 ± 0.20   3.9 ± .0.16 ConfidenceInterval 3.7 to 4.5 3.6 to 4.2 2 20 3.0 0.005  4.0 ± 0.11  3.8 ± 0.22Confidence Interval 3.8 to 4.3 3.4 to 4.3 Core Length (cm) 1 20 2.60.011  6.4 ± 0.41   6.0 ± .0.65 Confidence Interval 5.6 to 7.3 4.6 to7.4 2 20 16.5 0.000  8.7 ± 0.34  7.0 ± 0.29 Confidence Interval   8 to9.5 6.5 to 7.7 Leaf Length (cm) 1 20 12.6 0.000 30.9 ± 0.80 27.8 ± 0.74Confidence Interval 29.3 to 32.6 26.3 to 29.4 2 20 12.5 0.000 30.2 ±0.95 26.7 ± 0.80 Confidence Interval 28.2 to 32.2   25 to 28.4 LeafWidth (cm) 1 20 3.5 0.001 18.2 ± 0.44 18.9 ± 0.64 Confidence Interval17.3 to 19.2 17.6 to 20.2 2 20 6.8 0.000 15.7 ± 057  17.0 ± 0.68Confidence Interval 14.5 to 16.9 15.6 to 18.5 p[t*] statically differentat the 95% confidence level.

Statistical Analysis Measurable characteristics were assessed in severallocalities or dates and the results were analyzed separately. Unlessotherwise indicated, the statistical analyses were performed usingT-test. The results presented in actual t-value and probability valuesp[t] and are statically different at the 95% confidence level. Thestandard of deviation for each variety in the comparisons is presented.The confidence intervals were calculated based on [CI=mean±(SD×SE)].

‘CVX-35’ is most similar to cv. Bondi, however, in average ‘CVX-35’ issignificantly heavier in plant weight (gm) than cv. Bondi.

‘CVX-35’ is most similar to cv. Bondi; however, in average ‘CVX-35’ hasa longer core length (cm) than cv. Bondi.

‘CVX-35’ is most similar to cv. Bondi; however, in average ‘CVX-35’ hasa longer diameter (cm) than cv. Bondi.

‘CVX-35’ is most similar to cv. Bondi; however, in average ‘CVX-35’ hasa longer leaf length (cm) than cv. Bondi.

‘CVX-35’ is most similar to cv. Bondi; however, in average ‘CVX-35’ hasa smaller leaf width (cm) than cv. Bondi.

FURTHER EMBODIMENTS OF THE INVENTION

This invention is also directed to methods for producing a lettuce plantby crossing a first parent lettuce plant with a second parent lettuceplant, wherein the first parent lettuce plant or second parent lettuceplant is the lettuce plant from cultivar CVX-35. Further, both the firstparent lettuce plant and second parent lettuce plant may be fromcultivar CVX-35. Therefore, any methods using lettuce cultivar CVX-35are part of this invention, such as selfing, backcrosses, hybridbreeding, and crosses to populations. Plants produced using lettucecultivar CVX-35 as at least one parent are within the scope of thisinvention.

In one aspect of the invention, methods for developing novel plant typesare presented. In one embodiment the specific type of breeding method ispedigree selection, where both single plant selection and mass selectionpractices are employed. Pedigree selection, also known as the “Vilmorinsystem of selection,” is described in Fehr, Walter; Principles ofCultivar Development, Volume I, Macmillan Publishing Co., which ishereby incorporated by reference.

In lettuce breeding, lines may be selected for certain desiredappropriate characteristics. To optimize crossing, it is important tonote that lettuce is an obligate self-pollinating species. This meansthat the pollen is shed before stigma emergence, assuring 100%self-fertilization. Since each lettuce flower is an aggregate of about10-20 individual florets (typical of the Compositae family), removal ofthe anther tubes containing the pollen is performed by procedures wellknown in the art of lettuce breeding.

In one embodiment, the pedigree method of breeding is practiced whereselection is first practiced among F₂ plants. In the next season, themost desirable F₃ lines are first identified, and then desirable F₃plants within each line are selected. The following season and in allsubsequent generations of inbreeding, the most desirable families areidentified first, then desirable lines within the selected families arechosen, and finally desirable plants within selected lines are harvestedindividually. A family refers to lines that were derived from plantsselected from the same progeny row the preceding generation.

Using this pedigree method, two parents may be crossed using anemasculated female and a pollen donor (male) to produce F₁ offspring. Tooptimize crossing, it is important to note that lettuce is an obligateself-pollinating species. This means that the pollen is shed beforestigma emergence, assuring 100% self-fertilization. Since each lettuceflower is an aggregate of about 10-20 individual florets, manual removalof the anther tubes containing the pollen is tedious. As such, methodsof removing pollen well known to one of skill in the art, such asmisting to wash the pollen off prior to fertilization, may be employedto assure crossing or hybridization. The F₁ may be self-pollinated toproduce a segregating F₂ generation. Individual plants may then beselected which represent the desired phenotype in each generation (F₃,F₄, F₅, etc.) until the traits are homozygous or fixed within a breedingpopulation.

In addition to crossing, selection may be used to identify and isolatenew lettuce lines. In lettuce selection, lettuce seeds are planted, theplants are grown and single plant selections are made of plants withdesired characteristics. Seed from the single plant selections may beharvested, separated from seeds of the other plants in the field andre-planted. The plants from the selected seed may be monitored todetermine if they exhibit the desired characteristics of the originallyselected line. Selection work is preferably continued over multiplegenerations to increase the uniformity of the new line.

Choice of breeding or selection methods depends on the mode of plantreproduction, the heritability of the trait(s) being improved, and thetype of cultivar used commercially (e.g., F₁ hybrid cultivar, purelinecultivar, etc.). For highly heritable traits, a choice of superiorindividual plants evaluated at a single location will be effective,whereas for traits with low heritability, selection should be based onmean values obtained from replicated evaluations of families of relatedplants. Popular selection methods commonly include pedigree selection,modified pedigree selection, mass selection, and recurrent selection.

The complexity of inheritance influences choice of the breeding method.Backcross breeding may be used to transfer one or a few favorable genesfor a highly heritable trait into a desirable cultivar. This approachhas been used extensively for breeding disease-resistant cultivars.Various recurrent selection techniques are used to improvequantitatively inherited traits controlled by numerous genes. The use ofrecurrent selection in self-pollinating crops depends on the ease ofpollination, the frequency of successful hybrids from each pollination,and the number of hybrid offspring from each successful cross.

Each breeding program may include a periodic, objective evaluation ofthe efficiency of the breeding procedure. Evaluation criteria varydepending on the goal and objectives, but should include gain fromselection per year based on comparisons to an appropriate standard, theoverall value of the advanced breeding lines, and the number ofsuccessful cultivars produced per unit of input (e.g., per year, perdollar expended, etc.).

In one embodiment, promising advanced breeding lines are thoroughlytested and compared to appropriate standards in environmentsrepresentative of the commercial target area(s). The best lines arecandidates for new commercial cultivars; those still deficient in a fewtraits are used as parents to produce new populations for furtherselection.

These processes, which lead to the final step of marketing anddistribution, usually take several years from the time the first crossor selection is made. Therefore, development of new cultivars is atime-consuming process that requires precise forward planning, efficientuse of resources, and a minimum of changes in direction.

A most difficult task is the identification of individuals that aregenetically superior, because for most traits the true genotypic valueis masked by other confounding plant traits or environmental factors.One method of identifying a superior plant is to observe its performancerelative to other experimental plants and to a widely grown standardcultivar. If a single observation is inconclusive, replicatedobservations provide a better estimate of its genetic worth.

The goal of lettuce plant breeding is to develop new, unique andsuperior lettuce cultivars. In one embodiment, the breeder initiallyselects and crosses two or more parental lines, followed by repeatedselfing and selection, producing many new genetic combinations. Thebreeder can theoretically generate billions of different geneticcombinations via crossing, selfing and mutations. Preferably, each yearthe plant breeder selects the germplasm to advance to the nextgeneration. This germplasm may be grown under different geographical,climatic and soil conditions, and further selections are then made,during and at the end of the growing season.

In a preferred embodiment, the development of commercial lettucecultivars requires the development of lettuce varieties, the crossing ofthese varieties, and the evaluation of the crosses. Pedigree breedingand recurrent selection breeding methods may be used to developcultivars from breeding populations. Breeding programs may combinedesirable traits from two or more varieties or various broad-basedsources into breeding pools from which cultivars are developed byselfing and selection of desired phenotypes. The new cultivars may becrossed with other varieties and the hybrids from these crosses areevaluated to determine which have commercial potential.

Pedigree breeding is used commonly for the improvement ofself-pollinating crops or inbred lines of cross-pollinating crops. Twoparents which possess favorable, complementary traits are crossed toproduce an F₁. An F₂ population is produced by selfing one or severalF₁'s or by intercrossing two F₁'s (sib mating). Selection of the bestindividuals is usually begun in the F₂ population; then, beginning inthe F₃, the best individuals in the best families are usually selected.Replicated testing of families, or hybrid combinations involvingindividuals of these families, often follows in the F₄ generation toimprove the effectiveness of selection for traits with low heritability.At an advanced stage of inbreeding (e.g., F₆ and F₇), the best lines ormixtures of phenotypically similar lines are tested for potentialrelease as new cultivars.

Mass and recurrent selections can be used to improve populations ofeither self- or cross-pollinating crops. A genetically variablepopulation of heterozygous individuals may be identified or created byintercrossing several different parents. The best plants may be selectedbased on individual superiority, outstanding progeny, or excellentcombining ability. Preferably, the selected plants are intercrossed toproduce a new population in which further cycles of selection arecontinued.

Backcross breeding has been used to transfer genes for a simplyinherited, highly heritable trait into a desirable homozygous cultivaror line that is the recurrent parent. The source of the trait to betransferred is called the donor parent. The resulting plant is expectedto have the attributes of the recurrent parent (e.g., cultivar) and thedesirable trait transferred from the donor parent. After the initialcross, individuals possessing the phenotype of the donor parent may beselected and repeatedly crossed (backcrossed) to the recurrent parent.The resulting plant is expected to have the attributes of the recurrentparent (e.g., cultivar) and the desirable trait transferred from thedonor parent.

The single-seed descent procedure refers to planting a segregatingpopulation, harvesting a sample of one seed per plant, and using theone-seed sample to plant the next generation. When the population hasbeen advanced from the F₂ to the desired level of inbreeding, the plantsfrom which lines are derived will each trace to different F₂individuals. The number of plants in a population declines eachgeneration due to failure of some seeds to germinate or some plants toproduce at least one seed. As a result, not all of the F₂ plantsoriginally sampled in the population will be represented by a progenywhen generation advance is completed.

In addition to phenotypic observations, the genotype of a plant can alsobe examined. There are many laboratory-based techniques available forthe analysis, comparison and characterization of plant genotype; amongthese are Isozyme Electrophoresis, Restriction Fragment LengthPolymorphisms (RFLPs), Randomly Amplified Polymorphic DNAs (RAPDs),Arbitrarily Primed Polymerase Chain Reaction (AP-PCR), DNA AmplificationFingerprinting (DAF), Sequence Characterized Amplified Regions (SCARs),Amplified Fragment Length polymorphisms (AFLPs), Simple Sequence Repeats(SSRs—which are also referred to as Microsatellites), and SingleNucleotide Polymorphisms (SNPs).

Isozyme Electrophoresis and RFLPs have been widely used to determinegenetic composition. Shoemaker and Olsen, (Molecular Linkage Map ofSoybean (Glycine max) p 6.131-6.138 in S. J. O'Brien (ed) Genetic Maps:Locus Maps of Complex Genomes, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., (1993)) developed a molecular genetic linkage mapthat consisted of 25 linkage groups with about 365 RFLP, 11 RAPD, threeclassical markers and four isozyme loci. See also, Shoemaker, R. C.,RFLP Map of Soybean, p 299-309, in Phillips, R. L. and Vasil, I. K.,eds. DNA-Based Markers in Plants, Kluwer Academic Press, Dordrecht, theNetherlands (1994).

SSR technology is currently the most efficient and practical markertechnology; more marker loci can be routinely used and more alleles permarker locus can be found using SSRs in comparison to RFLPs. Forexample, Diwan and Cregan described a highly polymorphic microsatellitelocus in soybean with as many as 26 alleles. (Diwan, N. and Cregan, P.B., Theor. Appl. Genet. 95:22-225, 1997.) SNPs may also be used toidentify the unique genetic composition of the invention and progenyvarieties retaining that unique genetic composition. Various molecularmarker techniques may be used in combination to enhance overallresolution.

Molecular markers, which include markers identified through the use oftechniques such as Isozyme Electrophoresis, RFLPs, RAPDs, AP-PCR, DAF,SCARs, AFLPs, SSRs, and SNPs, may be used in plant breeding. One use ofmolecular markers is Quantitative Trait Loci (QTL) mapping. QTL mappingis the use of markers which are known to be closely linked to allelesthat have measurable effects on a quantitative trait. Selection in thebreeding process is based upon the accumulation of markers linked to thepositive effecting alleles and/or the elimination of the markers linkedto the negative effecting alleles from the plant's genome.

Molecular markers can also be used during the breeding process for theselection of qualitative traits. For example, markers closely linked toalleles or markers containing sequences within the actual alleles ofinterest can be used to select plants that contain the alleles ofinterest during a backcrossing breeding program. The markers can also beused to select toward the genome of the recurrent parent and against themarkers of the donor parent. This procedure attempts to minimize theamount of genome from the donor parent that remains in the selectedplants. It can also be used to reduce the number of crosses back to therecurrent parent needed in a backcrossing program. The use of molecularmarkers in the selection process is often called genetic marker enhancedselection or marker-assisted selection. Molecular markers may also beused to identify and exclude certain sources of germplasm as parentalvarieties or ancestors of a plant by providing a means of trackinggenetic profiles through crosses.

Mutation breeding is another method of introducing new traits intolettuce varieties. Mutations that occur spontaneously or areartificially induced can be useful sources of variability for a plantbreeder. The goal of artificial mutagenesis is to increase the rate ofmutation for a desired characteristic. Mutation rates can be increasedby many different means including temperature, long-term seed storage,tissue culture conditions, radiation (such as X-rays, Gamma rays,neutrons, Beta radiation, or ultraviolet radiation), chemical mutagens(such as base analogs like 5-bromo-uracil), antibiotics, alkylatingagents (such as sulfur mustards, nitrogen mustards, epoxides,ethyleneamines, sulfates, sulfonates, sulfones, or lactones), azide,hydroxylamine, nitrous acid or acridines. Once a desired trait isobserved through mutagenesis the trait may then be incorporated intoexisting germplasm by traditional breeding techniques. Details ofmutation breeding can be found in Principles of Cultivar Development byFehr, Macmillan Publishing Company, 1993.

The production of double haploids can also be used for the developmentof homozygous varieties in a breeding program. Double haploids areproduced by the doubling of a set of chromosomes from a heterozygousplant to produce a completely homozygous individual. For example, seeWan et al., Theor. Appl. Genet., 77:889-892, 1989.

Descriptions of other breeding methods that are commonly used fordifferent traits and crops can be found in one of several referencebooks (e.g., Principles of Plant Breeding John Wiley and Son, pp.115-161, 1960; Allard, 1960; Simmonds, 1979; Sneep et al., 1979; Fehr,1987; “Carrots and Related Vegetable Umbelliferae”, Rubatzky, V. E., etal., 1999).

Lettuce is an important and valuable vegetable crop. Thus, a continuinggoal of lettuce plant breeders is to develop stable, high yieldinglettuce cultivars that are agronomically sound. To accomplish this goal,the lettuce breeder preferably selects and develops lettuce plants withtraits that result in superior cultivars.

This invention also is directed to methods for producing a lettucecultivar plant by crossing a first parent lettuce plant with a secondparent lettuce plant wherein either the first or second parent lettuceplant is a lettuce plant of the line CVX-35. Further, both first andsecond parent lettuce plants can come from the cultivar CVX-35. Stillfurther, this invention also is directed to methods for producing acultivar CVX-35-derived lettuce plant by crossing cultivar CVX-35 with asecond lettuce plant and growing the progeny seed, and repeating thecrossing and growing steps with the cultivar CVX-35-derived plant from 0to 7 times. Thus, any such methods using the cultivar CVX-35 are part ofthis invention: selfing, backcrosses, hybrid production, crosses topopulations, and the like. All plants produced using cultivar CVX-35 asa parent are within the scope of this invention, including plantsderived from cultivar CVX-35. Advantageously, the cultivar is used incrosses with other, different, cultivars to produce first generation(F₁) lettuce seeds and plants with superior characteristics.

As used herein, the term plant includes plant cells, plant protoplasts,plant cell tissue cultures from which lettuce plants can be regenerated,plant calli, plant clumps and plant cells that are intact in plants orparts of plants, such as embryos, pollen, ovules, flowers, seeds, roots,anthers, and the like.

As is well known in the art, tissue culture of lettuce can be used forthe in vitro regeneration of a lettuce plant. Tissue culture of varioustissues of lettuces and regeneration of plants therefrom is well knownand widely published. For example, reference may be had to Teng et al.,HortScience. 1992, 27: 9, 1030-1032 Teng et al., HortScience. 1993, 28:6, 669-1671, Zhang et al., Journal of Genetics and Breeding. 1992, 46:3, 287-290, Webb et al., Plant Cell Tissue and Organ Culture. 1994, 38:1, 77-79, Curtis et al., Journal of Experimental Botany. 1994, 45: 279,1441-1449, Nagata et al., Journal for the American Society forHorticultural Science. 2000, 125: 6, 669-672. It is clear from theliterature that the state of the art is such that these methods ofobtaining plants are, and were, “conventional” in the sense that theyare routinely used and have a very high rate of success. Thus, anotheraspect of this invention is to provide cells which upon growth anddifferentiation produce lettuce plants having the physiological andmorphological characteristics of variety CVX-35.

With the advent of molecular biological techniques that have allowed theisolation and characterization of genes that encode specific proteinproducts, scientists in the field of plant biology developed a stronginterest in engineering the genome of plants to contain and expressforeign genes, or additional, or modified versions of native, orendogenous, genes (perhaps driven by different promoters) in order toalter the traits of a plant in a specific manner. Such foreignadditional and/or modified genes are referred to herein collectively astransgenes. Over the last fifteen to twenty years several methods forproducing transgenic plants have been developed, and the presentinvention, in particular embodiments, also relates to transformedversions of the claimed line.

Plant transformation preferably involves the construction of anexpression vector that will function in plant cells. Such a vector maycomprise DNA comprising a gene under control of or operatively linked toa regulatory element (for example, a promoter). The expression vectormay contain one or more such operably linked gene/regulatory elementcombinations. The vector(s) may be in the form of a plasmid, and can beused alone or in combination with other plasmids, to provide transformedlettuce plants, using transformation methods as described below toincorporate transgenes into the genetic material of the lettuceplant(s).

Expression Vectors for Lettuce Transformation Marker Genes

Expression vectors include at least one genetic marker, operably linkedto a regulatory element (a promoter, for example) that allowstransformed cells containing the marker to be either recovered bynegative selection, i.e., inhibiting growth of cells that do not containthe selectable marker gene, or by positive selection, i.e., screeningfor the product encoded by the genetic marker. Many commonly usedselectable marker genes for plant transformation are well known in thetransformation arts, and include, for example, genes that code forenzymes that metabolically detoxify a selective chemical agent which maybe an antibiotic or a herbicide, or genes that encode an altered targetwhich is insensitive to the inhibitor. A few positive selection methodsare also known in the art.

One commonly used selectable marker gene for plant transformation is theneomycin phosphotransferase II (nptII) gene, isolated from transposonTn5, which when placed under the control of plant regulatory signalsconfers resistance to kanamycin. Fraley et al., Proc. Natl. Acad. Sci.U.S.A., 80:4803 (1983). Another commonly used selectable marker gene isthe hygromycin phosphotransferase gene which confers resistance to theantibiotic hygromycin. Vanden Elzen et al., Plant Mol. Biol., 5:299(1985).

Additional selectable marker genes of bacterial origin that conferresistance to antibiotics include gentamycin acetyl transferase,streptomycin phosphotransferase, aminoglycoside-3′-adenyl transferase,the bleomycin resistance determinant. Hayford et al., Plant Physiol.86:1216 (1988), Jones et al., Mol. Gen. Genet., 210:86 (1987), Svab etal., Plant Mol. Biol. 14:197 (1990<Hille et al., Plant Mol. Biol. 7:171(1986). Other selectable marker genes confer resistance to herbicidessuch as glyphosate, glufosinate or broxynil. Comai et al., Nature317:741-744 (1985), Gordon-Kamm et al., Plant Cell 2:603-618 (1990) andStalker et al., Science 242:419-423 (1988).

Other selectable marker genes for plant transformation are not ofbacterial origin. These genes include, for example, mouse dihydrofolatereductase, plant 5-enolpyruvylshikimate-3-phosphate synthase and plantacetolactate synthase. Eichholtz et al., Somatic Cell Mol. Genet. 13:67(1987), Shah et al., Science 233:478 (1986), Charest et al., Plant CellRep. 8:643 (1990).

Another class of marker genes for plant transformation requiresscreening of presumptively transformed plant cells rather than directgenetic selection of transformed cells for resistance to a toxicsubstance such as an antibiotic. These genes are particularly useful toquantify or visualize the spatial pattern of expression of a gene inspecific tissues and are frequently referred to as reporter genesbecause they can be fused to a gene or gene regulatory sequence for theinvestigation of gene expression. Commonly used genes for screeningpresumptively transformed cells include .beta.-glucuronidase (GUS),.beta.-galaetesidase, luciferase and chloramphenicol, acetyltransferase.Jefferson, R. A., Plant Mol. Biol. Rep. 5:387 (1987), Teen et al., EMBOJ. 8:343 (1989), Koncz et al., Proc. Natl. Acad. Sci U.S.A. 84:131(1987), DeBlock et al., EMBO J. 3:1681 (1984).

Recently, in vivo methods for visualizing GUS activity that do notrequire destruction of plant tissue have been made available. MolecularProbes publication 2908, Imagene Green.TM., p. 1-4 (1993) and Naleway etal., J. Cell Biol. 115:151a (1991). However, these in vivo methods forvisualizing GUS activity have not proven useful for recovery oftransformed cells because of low sensitivity, high fluorescentbackgrounds and limitations associated with the use of luciferase genesas selectable markers.

More recently, a gene encoding Green Fluorescent Protein (GFP) has beenutilized as a marker for gene expression in prokaryotic and eukaryoticcells. Chalfie et al., Science 263:802 (1994). GFP and mutants of GFPmay be used as screenable markers.

Promoters

Genes included in expression vectors preferably are driven by nucleotidesequence comprising a regulatory element, for example, a promoter.Several types of promoters are now well known in the transformationarts, as are other regulatory elements that can be used alone or incombination with promoters.

As used herein, promoter includes reference to a region of DNA upstreamfrom the start of transcription and involved in recognition and bindingof RNA polymerase and other proteins to initiate transcription. A “plantpromoter” is a promoter capable of initiating transcription in plantcells. Examples of promoters under developmental control includepromoters that preferentially initiate transcription in certain tissues,such as leaves, roots, seeds, fibers, xylem vessels, tracheids, orsclerenchyma. Such promoters are referred to as “tissue-preferred”.Promoters which initiate transcription only in certain tissue arereferred to as “tissue-specific”. A “cell type” specific promoterprimarily drives expression in certain cell types in one or more organs,for example, vascular cells in roots or leaves. An “inducible” promoteris a promoter which is under environmental control. Examples ofenvironmental conditions that may affect transcription by induciblepromoters include anaerobic conditions or the presence of light.Tissue-specific, tissue-preferred, cell type specific, and induciblepromoters constitute the class of “non-constitutive” promoters. A“constitutive promoter” is a promoter which is active under mostenvironmental conditions.

A. Inducible Promoters

An inducible promoter is operably linked to a gene for expression inlettuce. Optionally, the inducible promoter is operably linked to anucleotide sequence encoding a signal sequence which is operably linkedto a gene for expression in lettuce. With an inducible promoter the rateof transcription increases in response to an inducing agent. Anyinducible promoter can be used in the instant invention. See Ward etal., Plant Mol. Biol. 22:361-366 (1993). Exemplary inducible promotersinclude, but are not limited to, that from the ACEI system whichresponds to copper (Meft et al., PNAS 90:4567-4571 (1993)); In2 genefrom maize which responds to benzenesulfonamide herbicide safeners(Hershey et al., Mol. Gen Genetics 227:229-237 (1991) and Gatz et al.,Mol. Gen. Genetics 243:32-38 (1994)) or Tet repressor from Tn10 (Gatz etal., Mol. Gen. Genetics 227:229-237 (1991). A particularly preferredinducible promoter is a promoter that responds to an inducing agent towhich plants do not normally respond. An exemplary inducible promoter isthe inducible promoter from a steroid hormone gene, the transcriptionalactivity of which is induced by a glucocorticosteroid hormone. Schena etal., Proc. Natl. Acad. Sci. U.S.A. 88:0421 (1991).

B. Constitutive Promoters

A constitutive promoter may be operably linked to a gene for expressionin lettuce or the constitutive promoter may operably linked to anucleotide sequence encoding a signal sequence which is operably linkedto a gene for expression in lettuce.

Many different constitutive promoters can be utilized in the instantinvention. Exemplary constitutive promoters include, but are not limitedto, the promoters from plant viruses such as the 35S promoter from CaMV(Odell et al., Nature 313:810-812 (1985) and the promoters from suchgenes as rice actin (McElroy et al., Plant Cell 2:163-171 (1990));ubiquitin (Christensen et al., Plant Mol. Biol. 12:619-632 (1989) andChristensen et al., Plant Mol. Biol. 18:675-689 (1992)); pEMU (Last etal., Theor. Appl. Genet. 81:581-588 (1991)); MAS (Velten et al., EMBO J.3:2723-2730 (1984)) and maize H3 histone (Lepetit et al., Mol. Gen.Genetics 231:276-285 (1992) and Atanassova et al., Plant Journal 2 (3):291-300 (1992)). The ALS promoter, Xbal/Ncol fragment 5′ to the Brassicanapus ALS3 structural gene (or a nucleotide sequence similarity to saidXbal/Ncol fragment), represents a particularly useful constitutivepromoter. See PCT application WO96/30530.

C. Tissue-Specific or Tissue Preferred Promoters

A tissue-specific promoter may be operably linked to a gene forexpression in lettuce. Optionally, the tissue-specific promoter isoperably linked to a nucleotide sequence encoding a signal sequencewhich is operably linked to a gene for expression in lettuce. Plantstransformed with a gene of interest operably linked to a tissue-specificpromoter produce the protein product of the transgene exclusively, orpreferentially, in a specific tissue.

Any tissue-specific or tissue-preferred promoter can be utilized in theinstant invention. Exemplary tissue-specific or tissue-preferredpromoters include, but are not limited to, a root-preferred promoter,such as that from the phaseolin gene (Murai et al., Science 23:476-482(1983) and Sengupta-Gopalan et al., Proc. Natl. Acad. Sci. U.S.A.82:3320-3324 (1985)); a leaf-specific and light-induced promoter such asthat from cab or rubisco (Simpson et al., EMBO J. 4(11):2723-2729 (1985)and Timko et al., Nature 318:579-582 (1985)); an anther-specificpromoter such as that from LAT52 (Twell et al., Mol. Gen. Genetics217:240-245 (1989)); a pollen-specific promoter such as that from Zm13(Guerrero et al., Mol. Gen. Genetics 244:161-168 (1993)) or amicrospore-preferred promoter such as that from apg (Twell et al., Sex.Plant Reprod. 6:217-224 (1993).

Signal Sequences for Targeting Proteins to Subcellular Compartments

Transport of protein produced by transgenes to a subcellular compartmentsuch as the chloroplast, vacuole, peroxisome, glyoxysome, cell wall ormitochondroin or for secretion into the apoplast, is accomplished bymeans of operably linking the nucleotide sequence encoding a signalsequence to the 5′ and/or 3′ region of a gene encoding the protein ofinterest. Targeting sequences at the 5′ and/or 3′ end of the structuralgene may determine, during protein synthesis and processing, where theencoded protein is ultimately compartmentalized.

The presence of a signal sequence directs a polypeptide to either anintracellular organelle or subcellular compartment or for secretion tothe apoplast. Many signal sequences are known in the art. See, forexample Becker et al., Plant Mol. Biol. 20:49 (1992), Close, P. S.,Master's Thesis, Iowa State University (1993), Knox, C., et al.,Structure and Organization of Two Divergent Alpha-Amylase Genes fromBarley, Plant Mol. Biol. 9:3-17 (1987), Lerner et al., Plant Physiol.91:124-129 (1989), Fontes et al., Plant Cell 3:483-496 (1991), Matsuokaet al., Proc. Natl. Acad. Sci. 88:834 (1991), Gould et al., J. Cell.Biol. 108:1657 (1989), Creissen et al., Plant J. 2:129 (1991), Kalderon,et al., A short amino acid sequence able to specify nuclear location,Cell 39:499-509 (1984), Steifel, et al., Expression of a maize cell wallhydroxyproline-rich glycoprotein gene in early leaf and root vasculardifferentiation, Plant Cell 2:785-793 (1990).

Foreign Protein Genes and Agronomic Genes

With transgenic plants according to the present invention, a foreignprotein can be produced in commercial quantities. Thus, techniques forthe selection and propagation of transformed plants, which are wellunderstood in the art, yield a plurality of transgenic plants that areharvested in a conventional manner, and a foreign protein then can beextracted from a tissue of interest or from total biomass. Proteinextraction from plant biomass can be accomplished by known methods whichare discussed, for example, by Heney and Orr, Anal. Biochem. 114:92-6(1981).

According to a preferred embodiment, the transgenic plant provided forcommercial production of foreign protein is lettuce. In anotherpreferred embodiment, the biomass of interest is seed. For transgenicplants that show higher levels of expression, a genetic map can begenerated, primarily via conventional RFLP, PCR and SSR analysis, whichidentifies the approximate chromosomal location of the integrated DNAmolecule. For exemplary methodologies in this regard, see Glick andThompson, Methods in Plant Molecular Biology and Biotechnology CRCPress, Boca Raton 269:284 (1993). Map information concerning chromosomallocation is useful for proprietary protection of a subject transgenicplant. If unauthorized propagation is undertaken and crosses made withother germplasm, the map of the integration region can be compared tosimilar maps for suspect plants, to determine if the latter have acommon parentage with the subject plant. Map comparisons may involvehybridizations, RFLP, PCR, SSR and sequencing, all of which areconventional techniques.

Likewise, by means of the present invention, agronomic genes can beexpressed in transformed plants. More particularly, plants can begenetically engineered to express various phenotypes of agronomicinterest. Exemplary genes implicated in this regard include, but are notlimited to, those categorized below:

1. Genes That Confer Resistance to Pests or Disease and That Encode:

A. Plant disease resistance genes. Plant defenses are often activated byspecific interaction between the product of a disease resistance gene(R) in the plant and the product of a corresponding avirulence (Avr)gene in the pathogen. A plant line can be transformed with clonedresistance gene to engineer plants that are resistant to specificpathogen strains. See, for example Jones et al., Science 266:789 (1994)(cloning of the tomato Cf-9 gene for resistance to Cladosporium fulvum);Martin et al., Science 262:1432 (1993) (tomato Pto gene for resistanceto Pseudomonas syringae pv. Tomato encodes a protein kinase); Mindrinoset al., Cell 78:1089 (1994) (Arabidopsis RSP2 gene for resistance toPseudomonas syringae).

B. A Bacillus thuringiensis protein, a derivative thereof or a syntheticpolypeptide modeled thereon. See, for example, Geiser et al., Gene48:109 (1986), who disclose the cloning and nucleotide sequence of a Btδ-endotoxin gene. Moreover, DNA molecules encoding δ-endotoxin genes canbe purchased from American Type Culture Collection, Manassas, Va., forexample, under ATCC Accession Nos. 40098, 67136, 31995 and 31998.

C. A lectin. See, for example, the disclosure by Van Damme et al., PlantMolec. Biol. 24:25 (1994), who disclose the nucleotide sequences ofseveral Clivia miniata mannose-binding lectin genes.

D. A vitamin-binding protein such as avidin. See PCT applicationUS93/06487, the contents of which are hereby incorporated by reference.The application teaches the use of avidin and avidin homologues aslarvicides against insect pests.

E. An enzyme inhibitor, for example, a protease or proteinase inhibitoror an amylase inhibitor. See, for example, Abe et al., J. Biol. Chem.262:16793 (1987) (nucleotide sequence of rice cysteine proteinaseinhibitor), Huub et al., Plant Molec. Biol. 21:985 (1993) (nucleotidesequence of cDNA encoding tobacco proteinase inhibitor I), Sumitani etal., Biosci. Biotoch. Biochem. 57:1243 (1993) (nucleotide sequence ofStreptomyces nitrosporeus α-amylase inhibitor).

F. An insect-specific hormone or pheromone such as an ecdysteroid andjuvenile hormone, a variant thereof, a mimetic based thereon, or anantagonist or agonist thereof. See, for example, the disclosure byHammock et al., Nature 344:458 (1990), of baculovirus expression ofcloned juvenile hormone esterase, an inactivator of juvenile hormone.

G. An insect-specific peptide or neuropeptide which, upon expression,disrupts the physiology of the affected pest. For example, see thedisclosures of Regan, J. Biol. Chem. 269:9 (1994) (expression cloningyields DNA coding for insect diuretic hormone receptor), and Pratt etal., Biochem. Biophys. Res. Comm. 163:1243 (1989) (an allostatin isidentified in Diploptera puntata). See also U.S. Pat. No. 5,266,317 toTomalski et al., who disclose genes encoding insect-specific, paralyticneurotoxins.

H. An insect-specific venom produced in nature by a snake, a wasp, etc.For example, see Pang et al., Gene 116:165 (1992), for disclosure ofheterologous expression in plants of a gene coding for a scorpioninsectotoxic peptide.

I. An enzyme responsible for a hyper accumulation of a monterpene, asesquiterpene, a steroid, hydroxamic acid, a phenylpropanoid derivativeor another non-protein molecule with insecticidal activity.

J. An enzyme involved in the modification, including thepost-translational modification, of a biologically active molecule; forexample, a glycolytic enzyme, a proteolytic enzyme, a lipolytic enzyme,a nuclease, a cyclase, a transaminase, an esterase, a hydrolase, aphosphatase, a kinase, a phosphorylase, a polymerase, an elastase, achitinase and a glucanase, whether natural or synthetic. See PCTapplication WO 93/02197 in the name of Scott et al., which discloses thenucleotide sequence of a callase gene. DNA molecules which containchitinase-encoding sequences can be obtained, for example, from the ATCCunder Accession Nos. 39637 and 67152. See also Kramer et al., InsectBiochem. Molec. Biol. 23:691 (1993), who teach the nucleotide sequenceof a cDNA encoding tobacco hookworm chitinase, and Kawalleck et al.,Plant Molec. Biol. 21:673 (1993), who provide the nucleotide sequence ofthe parsley ubi4-2 polyubiquitin gene.

K. A molecule that stimulates signal transduction. For example, see thedisclosure by Botella et al., Plant Molec. Biol. 24:757 (1994), ofnucleotide sequences for mung lettuce calmodulin cDNA clones, and Griesset al., Plant Physiol. 104:1467 (1994), who provide the nucleotidesequence of a maize calmodulin cDNA clone.

L. A hydrophobic moment peptide. See PCT application WO95/16776(disclosure of peptide derivatives of tachyolesin which inhibit fungalplant pathogens) and PCT application WO95/18855 (teaches syntheticantimicrobial peptides that confer disease resistance), the respectivecontents of which are hereby incorporated by reference.

M. A membrane permease, a channel former or a channel blocker. Forexample, see the disclosure of Jaynes et al., Plant Sci 89:43 (1993), ofheterologous expression of a cecropin-β, lytic peptide analog to rendertransgenic tobacco plants resistant to Pseudomonas solanacearum.

N. A viral-invasive protein or a complex toxin derived therefrom. Forexample, the accumulation of viral coat proteins in transformed plantcells imparts resistance to viral infection and/or disease developmenteffected by the virus from which the coat protein gene is derived, aswell as by related viruses. See Beachy et al., Ann. rev. Phytopathol.28:451 (1990). Coat protein-mediated resistance has been conferred upontransformed plants against alfalfa mosaic virus, cucumber mosaic virus,tobacco streak virus, potato virus X, potato virus Y, tobacco etchvirus, tobacco rattle virus and tobacco mosaic virus. Id.

O. An insect-specific antibody or an immunotoxin derived therefrom.Thus, an antibody targeted to a critical metabolic function in theinsect gut would inactivate an affected enzyme, killing the insect. Cf.Taylor et al., Abstract #497, Seventh Int'l Symposium on MolecularPlant-Microbe Interactions (Edinburgh, Scotland) (1994) (enzymaticinactivation in transgenic tobacco via production of single-chainantibody fragments).

P. A virus-specific antibody. See, for example, Tavladoraki et al.,Nature 366:469 (1993), who show that transgenic plants expressingrecombinant antibody genes are protected from virus attack.

Q. A developmental-arrestive protein produced in nature by a pathogen ora parasite. Thus, fungal endo-α-1, 4-D-polygalacturonases facilitatefungal colonization and plant nutrient release by solubilizing plantcell wall homo-α-1, 4-D-galacturonase. See Lamb at al., Bio/Technology10:1436 (1992). The cloning and characterization of a gene which encodesa lettuce endopolygalacturonase-inhibiting protein is described byToubart et al., Plant J. 2:367 (1992).

R. A development-arrestive protein produced in nature by a plant. Forexample, Logemann et al., Bioi/Technology 10:305 (1992), have shown thattransgenic plants expressing the barley ribosome-inactivating gene havean increased resistance to fungal disease.

S. A lettuce mosaic potyvirus (LMV) coat protein gene introduced intoLactuca sativa in order to increase its resistance to LMV infection. SeeDinant et al., Molecular Breeding. 1997, 3: 1, 75-86.

2. Genes that Confer Resistance to an Herbicide, for Example:

A. A herbicide that inhibits the growing point or meristem, such as animidazalinone or a sulfonylurea. Exemplary genes in this category codefor mutant ALS and AHAS enzyme as described, for example, by Lee et al.,EMBO J. 7:1241 (1988), and Miki et al., Theor. Appl. Genet. 80:449(1990), respectively.

B. Glyphosate (resistance impaired by mutant5-enolpyruvl-3-phosphikimate synthase (EPSP) and aroA genes,respectively) and other phosphono compounds such as glufosinate(phosphinothricin acetyl transferase, PAT and Streptomyces hygroscopicusphosphinothricin-acetyl transferase PAT bar genes), and pyridinoxy orphenoxy propionic acids and cycloshexones (ACCase inhibitor-encodinggenes). See, for example, U.S. Pat. No. 4,940,835 to Shah, et al., whichdiscloses the nucleotide sequence of a form of EPSP which can conferglyphosate resistance. A DNA molecule encoding a mutant aroA gene can beobtained under ATCC accession number 39256, and the nucleotide sequenceof the mutant gene is disclosed in U.S. Pat. No. 4,769,061 to Comai. Seealso Umaballava-Mobapathie in Transgenic Research. 1999, 8: 1, 33-44that discloses Lactuca sativa resistant to glufosinate. European patentapplication No. 0 333 033 to Kumada at al., and U.S. Pat. No. 4,975,374to Goodman et al., disclose nucleotide sequences of glutamine synthetasegenes which confer resistance to herbicides such as L-phosphinothricin.The nucleotide sequence of a phosphinothricin-acetyl-transferase gene isprovided in European application No. 0 242 246 to Leemans et al.,DeGreef et al., Bio/Technology 7:61 (1989), describe the production oftransgenic plants that express chimeric bar genes coding forphosphinothricin acetyl transferase activity. Exemplary of genesconferring resistance to phenoxy propionic acids and cycloshexones, suchas sethoxydim and haloxyfop are the Accl-S1, Accl-S2 and Accl-S3 genesdescribed by Marshall et al., Theor. Appl. Genet. 83:435 (1992).

C. A herbicide that inhibits photosynthesis, such as a triazine (psbAand gs+ genes) and a benzonitrile (nitrilase gene). Przibilla et al.,Plant Cell 3:169 (1991), describe the transformation of Chlamydomonaswith plasmids encoding mutant psbA genes. Nucleotide sequences fornitrilase genes are disclosed in U.S. Pat. No. 4,810,648 to Stalker, andDNA molecules containing these genes are available under ATCC AccessionNos. 53435, 67441, and 67442. Cloning and expression of DNA coding for aglutathione S-transferase is described by Hayes et al., Biochem. J.285:173 (1992).

D. Acetohydroxy acid synthase, which has been found to make plants thatexpress this enzyme resistant to multiple types of herbicides, has beenintroduced into a variety of plants. See Hattori et al., Mol. Gen.Genet. 246:419, 1995. Other genes that confer tolerance to herbicidesinclude a gene encoding a chimeric protein of rat cytochrome P4507A1 andyeast NADPH-cytochrome P450 oxidoreductase (Shiota et al., PlantPhysiol., 106:17, 1994), genes for glutathione reductase and superoxidedismutase (Aono et al., Plant Cell Physiol. 36:1687, 1995), and genesfor various phosphotransferases (Datta et al., Plant Mol. Biol. 20:619,1992).

E. Protoporphyrinogen oxidase (protox) is necessary for the productionof chlorophyll, which is necessary for all plant survival. The protoxenzyme serves as the target for a variety of herbicidal compounds. Theseherbicides also inhibit growth of all the different species of plantspresent, causing their total destruction. The development of plantscontaining altered protox activity which are resistant to theseherbicides are described in U.S. Pat. Nos. 6,288,306; 6,282,837;5,767,373; and international publication WO 01/12825.

3. Genes That Confer or Contribute to a Value-Added Trait, Such as:

A. Increased iron content of the lettuce, for example by transforming aplant with a soybean ferritin gene as described in Goto et al., ActaHorticulturae. 2000, 521, 101-109. Parallel to the improved iron contentenhanced growth of transgenic lettuces was also observed in earlydevelopment stages.

B. Decreased nitrate content of leaves, for example by transforming alettuce with a gene coding for a nitrate reductase. See for exampleCurtis et al., Plant Cell Report. 1999, 18: 11, 889-896.

C. Increased sweetness of the lettuce by transferring a gene coding formonellin that elicits a flavor sweeter than sugar on a molar basis. SeePenarrubia et al., Biotechnology. 1992, 10: 5, 561-564.

D. Modified fatty acid metabolism, for example, by transforming a plantwith an antisense gene of stearyl-ACP desaturase to increase stearicacid content of the plant. See Knultzon et al., Proc. Natl. Acad. Sci.USA 89:2625 (1992).

E. Modified carbohydrate composition effected, for example, bytransforming plants with a gene coding for an enzyme that alters thebranching pattern of starch. See Shiroza et al., J. Bacteriol. 170:810(1988) (nucleotide sequence of Streptococcus mutantsfructosyltransferase gene), Steinmetz et al., Mol. Gen. Genet. 20:220(1985) (nucleotide sequence of Bacillus subtilis levansucrase gene), Penet al., Bio/Technology 10:292 (1992) (production of transgenic plantsthat express Bacillus licheniformis .alpha.-amylase), Elliot et al.,Plant Molec. Biol. 21:515 (1993) (nucleotide sequences of tomatoinvertase genes), Sogaard et al., J. Biol. Chem. 268:22480 (1993)(site-directed mutagenesis of barley .alpha.-amylase gene), and Fisheret al., Plant Physiol. 102:1045 (1993) (maize endosperm starch branchingenzyme II).

4. Genes that Control Male-Sterility

A. Introduction of a deacetylase gene under the control of atapetum-specific promoter and with the application of the chemicalN-Ac-PPT. See international publication WO 01/29237.

B. Introduction of various stamen-specific promoters. See internationalpublications WO 92/13956 and WO 92/13957.

C. Introduction of the barnase and the barstar genes. See Paul et al.,Plant Mol. Biol. 19:611-622, 1992).

Methods for Lettuce Transformation

Numerous methods for plant transformation have been developed, includingbiological and physical, plant transformation protocols. See, forexample, Miki et al., “Procedures for Introducing Foreign DNA intoPlants” in Methods in Plant Molecular Biology and Biotechnology, GlickB. R. and Thompson, J. E. Eds. (CRC Press, Inc., Boca Raton, 1993) pages67-88. In addition, expression vectors and in vitro culture methods forplant cell or tissue transformation and regeneration of plants areavailable. See, for example, Gruber et al., “Vectors for PlantTransformation” in Methods in Plant Molecular Biology and Biotechnology,Glick B. R. and Thompson, J. E. Eds. (CRC Press, Inc., Boca Raton, 1993)pages 89-119.

A. Agrobacterium-Mediated Transformation

One method for introducing an expression vector into plants is based onthe natural transformation system of Agrobacterium. See, for example,Horsch et al., Science 227:1229 (1985). Curtis et al., Journal ofExperimental Botany. 1994, 45: 279, 1441-1449, Torres et al., Plant cellTissue and Organic Culture. 1993, 34: 3, 279-285, Dinant et al.,Molecular Breeding. 1997, 3: 1, 75-86. A. tumefaciens and A. rhizogenesare plant pathogenic soil bacteria which genetically transform plantcells. The Ti and Ri plasmids of A. tumefaciens and A. rhizogenes,respectively, carry genes responsible for genetic transformation of theplant. See, for example, Kado, C. I., Crit. Rev. Plant Sci. 10:1 (1991).Descriptions of Agrobacterium vector systems and methods forAgrobacterium-mediated gene transfer are provided by Gruber et al.,supra, Miki et al., supra, and Moloney et al., Plant Cell Reports 8:238(1989). See also, U.S. Pat. No. 5,591,616 issued Jan. 7, 1997.

B. Direct Gene Transfer

Several methods of plant transformation collectively referred to asdirect gene transfer have been developed as an alternative toAgrobacterium-mediated transformation. A generally applicable method ofplant transformation is microprojectile-mediated transformation whereinDNA is carried on the surface of microprojectiles measuring 1 to 4 μm.The expression vector is introduced into plant tissues with a biolisticdevice that accelerates the microprojectiles to speeds of 300 to 600 m/swhich is sufficient to penetrate plant cell walls and membranes.Russell, D. R., et al. Pl. Cell. Rep. 12(3, January), 165-169 (1993),Aragao, F. J. L., et al. Plant Mol. Biol. 20(2, October), 357-359(1992), Aragao, F. J. L., et al. Pl. Cell. Rep. 12(9, July), 483-490(1993). Aragao, Theor. Appl. Genet. 93: 142-150 (1996), Kim, J.;Minamikawa, T. Plant Science 117: 131-138 (1996), Sanford et al., Part.Sci. Technol. 5:27 (1987), Sanford, J. C., Trends Biotech. 6:299 (1988),Klein et al., Bio/Technology 6:559-563 (1988), Sanford, J. C., PhysiolPlant 7:206 (1990), Klein et al., Biotechnology 10:268 (1992).

Another method for physical delivery of DNA to plants is sonication oftarget cells. Zhang et al., Bio/Technology 9:996 (1991). Alternatively,liposome or spheroplast fusion has been used to introduce expressionvectors into plants. Deshayes et al., EMBO J., 4:2731 (1985), Christouet al., Proc Natl. Acad. Sci. U.S.A. 84:3962 (1987). Direct uptake ofDNA into protoplasts using CaCl₂ precipitation, polyvinyl alcohol orpoly-L-ornithine has also been reported. Hain et al., Mol. Gen. Genet.199:161 (1985) and Draper et al., Plant Cell Physiol. 23:451 (1982).Electroporation of protoplasts and whole cells and tissues have alsobeen described. Saker, M.; Kuhne, T. Biologia Plantarum 40(4): 507-514(1997/98), Donn et al., In Abstracts of VIIth International Congress onPlant Cell and Tissue Culture IAPTC, A2-38, p 53 (1990); D'Halluin etal., Plant Cell 4:1495-1505 (1992) and Spencer et al., Plant Mol. Biol.24:51-61 (1994). See also Chupean et al., Biotechnology. 1989, 7: 5,503-508.

Following transformation of lettuce target tissues, expression of theabove-described selectable marker genes allows for preferentialselection of transformed cells, tissues and/or plants, usingregeneration and selection methods now well known in the art.

The foregoing methods for transformation would typically be used forproducing a transgenic line. The transgenic line could then be crossed,with another (non-transformed or transformed) line, in order to producea new transgenic lettuce line. Alternatively, a genetic trait that hasbeen engineered into a particular lettuce cultivar using the foregoingtransformation techniques could be moved into another line usingtraditional backcrossing techniques that are well known in the plantbreeding arts. For example, a backcrossing approach could be used tomove an engineered trait from a public, non-elite inbred line into anelite inbred line, or from an inbred line containing a foreign gene inits genome into an inbred line or lines which do not contain that gene.As used herein, “crossing” can refer to a simple X by Y cross, or theprocess of backcrossing, depending on the context.

Gene Conversions

When the term lettuce plant, cultivar or lettuce line is used in thecontext of the present invention, this also includes any geneconversions of that line. The term gene converted plant as used hereinrefers to those lettuce plants which are developed by a plant breedingtechnique called backcrossing wherein essentially all of the desiredmorphological and physiological characteristics of a cultivar arerecovered in addition to the gene transferred into the line via thebackcrossing technique. Backcrossing methods can be used with thepresent invention to improve or introduce a characteristic into theline. The term backcrossing as used herein refers to the repeatedcrossing of a hybrid progeny back to one of the parental lettuce plantsfor that line. The parental lettuce plant that contributes the gene forthe desired characteristic is termed the nonrecurrent or donor parent.This terminology refers to the fact that the nonrecurrent parent is usedone time in the backcross protocol and therefore does not recur. Theparental lettuce plant to which the gene or genes from the nonrecurrentparent are transferred is known as the recurrent parent as it is usedfor several rounds in the backcrossing protocol (Poehlman & Sleper,1994; Fehr, 1987). In a typical backcross protocol, the originalcultivar of interest (recurrent parent) is crossed to a second line(nonrecurrent parent) that carries the single gene of interest to betransferred. The resulting progeny from this cross are then crossedagain to the recurrent parent and the process is repeated until alettuce plant is obtained wherein essentially all of the desiredmorphological and physiological characteristics of the recurrent parentare recovered in the converted plant, in addition to the singletransferred gene from the nonrecurrent parent.

The selection of a suitable recurrent parent is an important step for asuccessful backcrossing procedure. The goal of a backcross protocol isto alter or substitute traits or characteristics in the original line.To accomplish this, a gene or genes of the recurrent cultivar aremodified or substituted with the desired gene or genes from thenonrecurrent parent, while retaining essentially all of the rest of thedesired genetic, and therefore the desired physiological andmorphological, constitution of the original line. The choice of theparticular nonrecurrent parent will depend on the purpose of thebackcross. One of the major purposes is to add some commerciallydesirable, agronomically important trait or traits to the plant. Theexact backcrossing protocol will depend on the characteristics or traitsbeing altered to determine an appropriate testing protocol. Althoughbackcrossing methods are simplified when the characteristic beingtransferred is a dominant allele, a recessive allele may also betransferred. In this instance it may be necessary to introduce a test ofthe progeny to determine if the desired characteristic has beensuccessfully transferred.

Many gene traits have been identified that are not regularly selectedfor in the development of a new line but that can be improved bybackcrossing techniques. Gene traits may or may not be transgenic,examples of these traits include but are not limited to, herbicideresistance, resistance for bacterial, fungal, or viral disease, insectresistance, enhanced nutritional quality, industrial usage, yieldstability, yield enhancement, male sterility, modified fatty acidmetabolism, and modified carbohydrate metabolism. These genes aregenerally inherited through the nucleus. Several of these gene traitsare described in U.S. Pat. Nos. 5,777,196; 5,948,957 and 5,969,212, thedisclosures of which are specifically hereby incorporated by reference.

Tissue Culture

Further reproduction of the variety can occur by tissue culture andregeneration. Tissue culture of various tissues of lettuce andregeneration of plants therefrom is well known and widely published. Forexample, reference may be had to Teng et al., HortScience. 1992, 27: 9,1030-1032 Teng et al., HortScience. 1993, 28: 6, 669-1671, Zhang et al.,Journal of Genetics and Breeding. 1992, 46: 3, 287-290, Webb et al.,Plant Cell Tissue and Organ Culture. 1994, 38: 1, 77-79, Curtis et al.,Journal of Experimental Botany. 1994, 45: 279, 1441-1449, Nagata et al.,Journal for the American Society for Horticultural Science. 2000, 125:6, 669-672, and Ibrahim et al., Plant Cell, Tissue and Organ Culture.(1992), 28(2): 139-145. It is clear from the literature that the stateof the art is such that these methods of obtaining plants are routinelyused and have a very high rate of success. Thus, another aspect of thisinvention is to provide cells which upon growth and differentiationproduce lettuce plants having the physiological and morphologicalcharacteristics of cultivar CVX-35.

As used herein, the term “tissue culture” indicates a compositioncomprising isolated cells of the same or a different type or acollection of such cells organized into parts of a plant. Exemplarytypes of tissue cultures are protoplasts, calli, meristematic cells, andplant cells that can generate tissue culture that are intact in plantsor parts of plants, such as leaves, pollen, embryos, roots, root tips,anthers, pistils, flowers, seeds, petioles, suckers and the like. Meansfor preparing and maintaining plant tissue culture are well known in theart. By way of example, a tissue culture comprising organs has been usedto produce regenerated plants. U.S. Pat. Nos. 5,959,185; 5,973,234 and5,977,445 describe certain techniques, the disclosures of which areincorporated herein by reference.

Additional Breeding Methods

This invention also is directed to methods for producing a lettuce plantby crossing a first parent lettuce plant with a second parent lettuceplant wherein the first or second parent lettuce plant is a lettuceplant of cultivar CVX-35. Further, both first and second parent lettuceplants can come from lettuce cultivar CVX-35. Thus, any such methodsusing lettuce cultivar CVX-35 are part of this invention: selfing,backcrosses, hybrid production, crosses to populations, and the like.All plants produced using lettuce cultivar CVX-35 as at least one parentare within the scope of this invention, including those developed fromcultivars derived from lettuce cultivar CVX-35. Advantageously, thislettuce cultivar could be used in crosses with other, different, lettuceplants to produce the first generation (F₁) lettuce hybrid seeds andplants with superior characteristics. The cultivar of the invention canalso be used for transformation where exogenous genes are introduced andexpressed by the cultivar of the invention. Genetic variants createdeither through traditional breeding methods using lettuce cultivarCVX-35 or through transformation of cultivar CVX-35 by any of a numberof protocols known to those of skill in the art are intended to bewithin the scope of this invention.

The following describes breeding methods that may be used with lettucecultivar CVX-35 in the development of further lettuce plants. One suchembodiment is a method for developing cultivar CVX-35 progeny lettuceplants in a lettuce plant breeding program comprising: obtaining thelettuce plant, or a part thereof, of cultivar CVX-35, utilizing saidplant or plant part as a source of breeding material, and selecting alettuce cultivar CVX-35 progeny plant with molecular markers in commonwith cultivar CVX-35 and/or with morphological and/or physiologicalcharacteristics selected from the characteristics listed in Table 1.Breeding steps that may be used in the lettuce plant breeding programinclude pedigree breeding, backcrossing, mutation breeding, andrecurrent selection. In conjunction with these steps, techniques such asRFLP-enhanced selection, genetic marker enhanced selection (for exampleSSR markers) and the making of double haploids may be utilized.

Another method which may be used involves producing a population oflettuce cultivar CVX-35-progeny lettuce plants, comprising crossingcultivar CVX-35 with another lettuce plant, thereby producing apopulation of lettuce plants, which, on average, derive 50% of theiralleles from lettuce cultivar CVX-35. A plant of this population may beselected and repeatedly selfed or sibbed with a lettuce cultivarresulting from these successive filial generations. One embodiment ofthis invention is the lettuce cultivar produced by this method and thathas obtained at least 50% of its alleles from lettuce cultivar CVX-35.

One of ordinary skill in the art of plant breeding would know how toevaluate the traits of two plant varieties to determine if there is nosignificant difference between the two traits expressed by thosevarieties. For example, see Fehr and Walt, Principles of CultivarDevelopment, p 261-286 (1987). Thus the invention includes lettucecultivar CVX-35 progeny lettuce plants comprising a combination of atleast two cultivar CVX-35 traits selected from the group consisting ofthose listed in Table 1 or the cultivar CVX-35 combination of traitslisted above, so that said progeny lettuce plant is not significantlydifferent for said traits than lettuce cultivar CVX-35 as determined atthe 5% significance level when grown in the same environmentalconditions. Using techniques described herein, molecular markers may beused to identify said progeny plant as a lettuce cultivar CVX-35 progenyplant. Mean trait values may be used to determine whether traitdifferences are significant, and preferably the traits are measured onplants grown under the same environmental conditions. Once such avariety is developed its value is substantial since it is important toadvance the germplasm base as a whole in order to maintain or improvetraits such as yield, disease resistance, pest resistance, and plantperformance in extreme environmental conditions.

Progeny of lettuce cultivar CVX-35 may also be characterized throughtheir filial relationship with lettuce cultivar CVX-35, as for example,being within a certain number of breeding crosses of lettuce cultivarCVX-35. A breeding cross is a cross made to introduce new genetics intothe progeny, and is distinguished from a cross, such as a self or a sibcross, made to select among existing genetic alleles. The lower thenumber of breeding crosses in the pedigree, the closer the relationshipbetween lettuce cultivar CVX-35 and its progeny. For example, progenyproduced by the methods described herein may be within 1, 2, 3, 4 or 5breeding crosses of lettuce cultivar CVX-35.

The foregoing invention has been described in detail by way ofillustration and example for purposes of clarity and understanding.However, it will be obvious that certain changes and modifications suchas single gene modifications and mutations, somoclonal variants, variantindividuals selected from large populations of the plants of the instantvariety and the like may be practiced within the scope of the invention,as limited only by the scope of the appended claims.

Deposits

Applicant(s) will make a deposit of at least 2500 seeds of LettuceCultivar CVX-35 with the American Type Culture Collection (ATCC),Manassas, Va. 20110 USA, ATCC Deposit No. ______. The seeds depositedwith the ATCC on ______ will be taken from the deposit maintained byCentral Valley Seeds, 485 Victor Way, Suite 10, Salinas Calif. 93907since prior to the filing date of this application. Access to thisdeposit will be available during the pendency of the application to theCommissioner of Patents and Trademarks and persons determined by theCommissioner to be entitled thereto upon request. Upon issue of claims,the Applicant(s) will make available to the public, pursuant to 37 CFR1.808, a deposit of at least 2500 seeds of cultivar CVX-35 with theAmerican type Culture Collection (ATCC), 10801 University Boulevard,Manassas, Va. 20110-2209. This deposit of the lettuce cultivar CVX-35will be maintained in the ATCC depository, which is a public depository,for a period of 30 years, or 5 years after the most recent request, orfor the enforceable life of the patent, whichever is longer, and will bereplaced if it becomes nonviable during that period. Additionally,Applicants have or will satisfy all the requirements of 37 C.F.R. § §1.801-1.809, including providing an indication of the viability of thesample. Applicants have no authority to waive any restrictions imposedby law on the transfer of biological material or its transportation incommerce. Applicants do not waive any infringement of their rightsgranted under this patent or under the Plant Variety Protection Act (7USC 2321 et seq.).

1-26. (canceled) 27: A seed of lettuce designated CVX-35, wherein arepresentative sample of seed of said lettuce having been depositedunder ATCC Accession No. PTA-123786. 28: A lettuce plant, or a partthereof or a plant cell thereof, produced by growing the seed of claim1. 29: The lettuce part of claim 28, wherein the lettuce part isselected from the group consisting of: a leaf, a flower, a head, anovule, pollen and a cell. 30: A lettuce plant having all of thecharacteristics of lettuce CVX-35 listed in Table 1 when grown in thesame environmental condition, or a part or a plant cell thereof. 31: Atissue culture of regenerable cells produced from the plant or plantpart of claim 28, wherein cells of the tissue culture are produced froma plant part selected from the group consisting of protoplasts, embryos,meristematic cells, callus, pollen, ovules, flowers, seeds, leaves,roots, root tips, anthers, stems, petioles, cotyledons and hypocotylswherein a plant regenerated from the tissue culture has all of thecharacteristics of lettuce CVX-35 listed in Table 1 when grown in thesame environmental condition and wherein a representative sample of seedof said lettuce having been deposited under ATCC Accession No.PTA-123786. 32: A lettuce plant regenerated from the tissue culture ofclaim 31, said plant having the characteristics of lettuce CVX-35,wherein a representative sample of seed of said lettuce having beendeposited under ATCC Accession No. PTA-123786. 33: A lettuce headproduced from the plant of claim
 28. 34: A method for producing alettuce head comprising a) growing the lettuce plant of claim 28 toproduce a lettuce head, and b) harvesting said lettuce head. 35: Alettuce head produced by the method of claim
 34. 36: A method forproducing a lettuce seed comprising crossing a first parent lettuceplant with a second parent lettuce plant and harvesting the resultant F₁lettuce seed, wherein said first parent lettuce plant and/or secondparent lettuce plant is the lettuce plant of claim
 28. 37: An F₁ lettuceseed produced by the method of claim
 36. 38: The method of claim 36,wherein the method further comprises: (a) crossing a plant grown fromsaid F₁ lettuce seed with itself or a different lettuce plant to producea seed of a progeny plant of a subsequent generation; (b) growing aprogeny plant of a subsequent generation from said seed of a progenyplant of a subsequent generation and crossing the progeny plant of asubsequent generation with itself or a second plant to produce a progenyplant of a further subsequent generation; and (c) repeating steps (a)and (b) using said progeny plant of a further subsequent generation fromstep (b) in place of the plant grown from said F₁ lettuce seed in step(a), wherein steps (a) and (b) are repeated with sufficient inbreedingto produce an inbred lettuce plant derived from the lettuce CVX-35. 39:A method for producing a lettuce seed comprising self-pollinating thelettuce plant of claim 28 and harvesting the resultant lettuce seed. 40:An F₁ lettuce seed produced by the method of claim
 39. 41: A method ofproducing a lettuce plant derived from the lettuce CVX-35, the methodcomprising the steps of: (a) crossing the plant of claim 28 with asecond lettuce plant to produce a progeny plant; (b) crossing theprogeny plant of step (a) with itself or the second lettuce plant instep (a) to produce a seed; (c) growing a progeny plant of a subsequentgeneration from the seed produced in step (b); (d) crossing the progenyplant of a subsequent generation of step (c) with itself or the secondlettuce plant in step (a) to produce a lettuce plant derived from thelettuce CVX-35. 42: The method of claim 41 further comprising the stepof: (e) repeating step b) and/or c) for at least 1 more generation toproduce a lettuce plant derived from the lettuce CVX-35. 43: The plantor plant part of claim 28, wherein the plant further comprises at leastone locus conversion. 44: The plant of claim 43, wherein the locusconversion confers said plant with a trait selected from the groupconsisting of male sterility, male fertility, herbicide resistance,insect resistance, disease resistance, water stress tolerance, heattolerance, improved shelf life, delayed shelf life, and improvednutritional quality. 45: The plant of claim 44, wherein the locusconversion is an artificially mutated gene or nucleotide sequence. 46: Amethod of introducing a desired trait into lettuce CVX-35 comprising:(a) crossing a lettuce CVX-35 plant grown from lettuce CVX-35 seed,wherein a representative sample of seed has been deposited under ATCCAccession No. PTA-123786, with another lettuce plant that comprises adesired trait to produce F₁ progeny plants, wherein the desired trait isselected from the group consisting of insect resistance, herbicideresistance, disease resistance, water stress tolerance, heat tolerance,improved shelf life, delayed shelf life, and improved nutritionalquality; (b) selecting one or more progeny plants that have the desiredtrait to produce selected progeny plants; (c) crossing the selectedprogeny plants with the lettuce CVX-35 plants to produce backcrossprogeny plants; (d) selecting for backcross progeny plants that have thedesired trait and the physiological and morphological characteristics oflettuce CVX-35 listed in Table 1 when grown in the same environmentalcondition to produce selected backcross progeny plants; and (e)repeating steps (c) and (d) three or more times in succession to produceselected fourth or higher backcross progeny plants that comprise thedesired trait and the physiological and morphological characteristics oflettuce CVX-35 listed in Table 1 when grown in the same environmentalcondition. 47: A method for developing a lettuce plant in a lettuceplant breeding program, comprising applying plant breeding techniquescomprising recurrent selection, backcrossing, pedigree breeding, markerenhanced selection, or transformation to the lettuce plant of claim 28,or its parts, wherein application of said techniques results indevelopment of a lettuce plant.